INSTITUTE OF PHYSICS PUBLISHING JOURNAL OF PHYSICS: CONDENSED MATTER J. Phys.: Condens. Matter 14 (2002) R597–R624 PII: S0953-8984(02)21355-7 TOPICAL REVIEW Surface-enhanced Raman scattering and biophysics Katrin Kneipp1, Harald Kneipp, Irving Itzkan, Ramachandra R Dasari and Michael S Feld Physics Department, Technical University Berlin, D 10623 Berlin, Germany and G R Harrison Spectroscopy Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA E-mail: [email protected] Received 13 December 2001, in final form 5 April 2002 Published 26 April 2002 Online at stacks.iop.org/JPhysCM/14/R597 Abstract Surface-enhanced Raman scattering (SERS) is a spectroscopic technique which combines modern laser spectroscopy with the exciting optical properties of metallic nanostructures, resulting in strongly increased Raman signals when molecules are attached to nanometre-sized gold and silver structures. The effect provides the structural information content of Raman spectroscopy together with ultrasensitive detection limits, allowing Raman spectroscopy of single molecules. Since SERS takes place in the local fields of metallic nanostructures, the lateral resolution of the technique is determined by the confinement of the local fields, which can be two orders of magnitude better than the diffraction limit. Moreover, SERS is an analytical technique, which can give information on surface and interface processes. SERS opens up exciting opportunities in the field of biophysical and biomedical spectroscopy, where it provides ultrasensitive detection and characterization of biophysically/biomedically relevant molecules and processes as well as a vibrational spectroscopy with extremely high spatial resolution. The article briefly introduces the SERS effect and reviews contemporary SERS studies in biophysics/biochemistry and in life sciences. Potential and limitations of the technique are briefly discussed. Contents 1. Introduction 597 2. Raman scattering and surface-enhanced Raman scattering 599 2.1. SERS mechanisms and SERS enhancement factors 602 2.2. SERS-active substrates for biophysical applications 606 2.3. Anti-Stokes SERS—a two-photon Raman probe for biophysics 607 2.4. SERS—a single-molecule tool 609 1 Author to whom any correspondence should be addressed. 0953-8984/02/180597+28$30.00 © 2002 IOP Publishing Ltd Printed in the UK R597 R598 K Kneipp et al 3. Examples for application of SERS in biophysics, biochemistry and biomedicine 610 3.1. Detection and identification of neurotransmitters 611 3.2. SERS detection and identification of microorganisms 613 3.3. Immunoassays utilizing SERS 614 3.4. DNA and gene probes based on SERS labels 615 3.5. Probing DNA and DNA fragments without labelling based on their intrinsic Raman spectra 617 3.6. SERS studies inside living human cells 618 4. Summary, prospects and limitations of SERS in biophysics 620 References 621 1. Introduction Laser spectroscopic methods play an increasingly important role in biophysics/biochemistry and in life sciences. Application of spectroscopy includes both the field of basic research in biophysics/biochemistry and the development of new methods for diagnosis of disease and therapy control. Identification and structural characterization, including monitoring structural changes of molecules, play a central role in biophysical/biochemical spectroscopy. Therefore, vibrational spectroscopic techniques such as Raman spectroscopy, which provide high structural information content, are of particular interest. A great disadvantage in any application of Raman spectroscopy results from the extremely small cross section of the Raman process, which is 12–14 orders of magnitude below fluorescence cross sections. Therefore, in the 1970s, a discovery which showed unexpectedly high Raman signals from pyridine on a rough silver electrode attracted considerable attention [1], particularly after experiments in different laboratories gave evidence that the enormously strong Raman signal must be caused by a true enhancement of the Raman scattering (RS) efficiency itself and not by more scattering molecules [2, 3]. Within the next few years, strongly enhanced Raman signals were verified for many different molecules [4] which had been attached to various ‘rough’ metal surfaces, and the effect was named ‘surface-enhanced Raman scattering (SERS)’. For an overview see [5, 6]. The discovery showed promise to overcome the traditionally low sensitivity problem in Raman spectroscopy. Estimated enhancement factors of the Raman signal started with modest factors of 103–105 in the first reports on SERS. Later, many authors claimed enhancement factors of about 1010– 1011 for dye molecules in surface-enhanced resonance Raman (SERRS) experiments [7–13]. About 20 years after the discovery of the effect, new methods for determining cross sections effective in SERS resulted in unexpectedly large cross sections on the order of at least 10−16 cm2/molecule corresponding to enhancement factors of about 14 orders of magnitude compared with ‘normal’ non-resonant RS [14]. Such effective Raman cross sections, on the same order of magnitude as fluorescence cross sections of ‘good’ laser dyes, made RS a single-molecule tool [15–23]. Another generally interesting aspect of SERS is attributed to its spatial resolution. Exploiting local optical fields of special metallic nanostructures, SERS can provide lateral resolutions better than 10 nm [24–27], which is two orders of magnitude below the diffraction limit and even smaller than the resolution of common near field microscope tips. Since its early days, the SERS effect has also been particularly appealing in the field of biophysics and biochemistry for several reasons. Most exciting for biophysical studies might be the trace analytical capabilities of the SERS effect together with its high structural selectivity and the opportunity to measure Raman spectra from extremely small volumes. Particularly, single-molecule capabilities open up exciting SERS and biophysics R599 perspectives for SERS as a tool in laboratory medicine and for basic research in biophysics, where SERS can offer interesting new aspects compared with fluorescence, which is widely used as a single-molecule spectroscopy tool in biophysics. One of the most spectacular applications of single-molecule SERS might appear in the field of rapid DNA sequencing using the Raman spectroscopic characterization of specific DNA fragments down to structurally sensitive detection of single bases without the use of fluorescent or radioactive labels [18]. Interesting aspects in biophysical applications of SERS come also from exploiting SERRS. In addition to the increased cross sections, resonance Raman scattering (RRS) has the advan- tage of higher specificity, since the resonance Raman spectrum is dominated by molecular vibrations which are related to the part of the molecule responsible for the appropriate res- onant electronic transition. SERRS opens interesting opportunities for large biomolecules, where it allows a selective vibrational probe of the chromophoric systems such as chlorophyll, pheophytin and carotenoids [28–33]. As a further advantage, the fluorescence background, which can make RRS spectroscopy extremely difficult, has been quenched in many SERRS experiments by new nonradiative decay channels provided by the SERS-active metal. Another useful capability of SERS in the field of biophysics comes from the potential of the method for providing information on molecules residing on surfaces and on surface and interface processes. For example, ‘SERS-active’ silver or gold electrodes with a defined potential can be used as a model environment for studying biologically relevant processes, such as charge transfer transitions in cytochrome c [34, 35]. There are excellent reviews that summarize SERS studies on biological molecules in the 1980s and early 1990s [36–41]. Therefore, this article will mainly review SERS studies in biophysics and life sciences performed within the last half decade. After introducing today’s understanding of the SERS effect, we review contemporary SERS studies in that field. The potential and limitations of SERS in biophysics/biochemistry and in life sciences as a tool in biophysical and biomedical spectroscopy are considered. 2. Raman scattering and surface-enhanced Raman scattering Before discussing the effect of SERS, we briefly recall the Raman effect [42, 43]. In the ‘photon picture’, we consider the Raman effect as a scattering process between a photon and a molecule (see also figure 1(a)). Incident photons hνL are inelastically scattered from a molecule and shifted in frequency by the energy of its characteristic molecular vibrations hνM . Frequency-shifted scattered photons can occur at lower and higher energy relative to the incoming photons, depending on whether they interacted with a molecule in the vibrational ground state or an excited vibrational state. In the first case, photons lose energy by exciting a vibration and the scattered light appears at a lower frequency νS , called the Stokes scattering. By interacting with a molecule in an excited vibrational state, the photons gain energy from the molecular vibrations and the scattered signal appears at higher frequency νaS, called anti-Stokes scattering. In a ‘classical view’ of the Raman effect, light is an electromagnetic wave which induces a dipole moment in the molecule. The generation of this dipole moment by the incoming electric field is modulated by the molecular vibrations, since the polarizibility α is a function of the vibrational